SUMMARY. MarR (Multiple antibiotic resistance Repressor) proteins comprise an ancient family of transcription factors that are widely conserved in bacteria. In the enteric pathogen Salmonella Typhimurium, MarR is a negative regulator of the AcrAB-TolC drug efflux pump, while another MarR transcription factor called SlyA is a counter-silencer required for the expression of horizontally-acquired virulence genes. We have recently shown that despite its divergent function, SlyA shares with MarR the ability to undergo allosteric modulation by aromatic carboxylate molecules and can influence the mar phenotype by repressing the marRAB operon. Inactivation of TolC, an essential component of the AcrAB and other RND-family efflux pumps, phenocopies a slyA mutation, reducing the expression of Salmonella virulence genes. This suggests that efflux regulates the levels of an endogenous metabolite that interacts with SlyA, which in turn controls MarR expression, completing a regulatory circuit that coordinately links antimicrobial resistance, virulence, and bacterial metabolism. This proposal investigates the hypothesis that Salmonella antimicrobial resistance and virulence are coordinately regulated in response to metabolic signals by the MarR and SlyA transcription factors through the following specific aims: (1) Identification of endogenous ligand(s) that modulate MarR/SlyA activity. Preliminary data indicate that SlyA-interacting ligand(s) are aromatic carboxylates. Specific endogenous ligands will be identified by genetic and metabolomic methods and characterized with regard to affinity and allosteric inhibition. (2) Functional characterization of MarR-SlyA-TolC regulatory interactions in Salmonella antimicrobial resistance and virulence. The contribution of the MarR-SlyA regulatory network to Salmonella phenotypic antimicrobial resistance and virulence will be assessed in vitro and in vivo. (3) Comparative analysis of the MarR and SlyA regulons. MarR/SlyA-regulated genes and binding sites will be comprehensively identified to determine the regulatory requirements for repression and counter-silencing, respectively, and to elucidate the mechanisms of transcriptional network evolution. These studies will provide important insights into a central regulatory network linking bacterial virulence, drug resistance, and metabolism, which can lead to the identification of new therapeutic targets for the prevention or treatment of bacterial infections.